Transgenic animal model for alzheimer disease

ABSTRACT

Animal model involving transgenic manipulation of amyloid precursor protein, useful for testing potential therapeutic agents for the treatment of neurodegenerative disorders, in particular Alzheimer&#39;s disease.

[0001] The present invention relates to an animal model useful fortesting potential therapeutic agents for the treatment ofneurodegenerative disorders, in particular Alzheimer's disease (AD).

[0002] More particularly the invention relates to an animal modelinvolving transgenic manipulation of amyloid precursor protein (APP).

[0003] The lack of an experimental animal model for AD that reflects thepathological mechanisms is a major obstacle for both basic research anddrug development. As one approach to such models, reproduction ofcharacteristic lesions such as senile plaques, neurofibrillarypathology, and cell loss in certain areas of hippocampus and cortex canbe attempted. However, it is presently unclear whether these lesions arecause or consequence of the disease process. An alternative approach formodel generation is to use factors known to lead to the disease.Recently, genetic studies revealed mutations in APP, which cosegregatewith early onset of familial AD in the fifth or sixth decade of life andfollow an autosomal dominant inheritance pattern. Three distinctmissense mutations affect codon 717 of APP (altering V717→I {hereinafterreferred to as the London mutation}, V717→G and V717→F in thepolypeptide), while codons 670/671 (altering K670→N and M671→L in thepolypeptide, hereinafter referred to as the Swedish mutation) arealtered in the APP gene of a Swedish AD pedigree (numbers according toAPP770). These mutations flank the part of APP that gives rise to βA4,the principal component of the filaments deposited in plaques in thebrains of AD patients. In vitro studies have indicated that the Swedishmutation leads to increased formation of a soluble form of βA4, whilethe APP717 mutations gives rise to a higher proportion of a longer βA4variant which facilitates filament formation. Together with the findingthat filamentous βA4 is toxic in vitro, this suggests that the APPmutations may lead to AD via a mechanism involving βA4, but othermechanisms cannot be excluded.

[0004] More recently, transgenic mice have been generated, expressingAPP with mutations in codons 717 and 670/671, using severalneuron-specific promoters to drive expression of human APP cDNAs.Although protein levels reaching or exceeding the amount of endogenousAPP have been obtained, the full pattern of histological alterationscharacteristic of AD have not been seen in the transgenic mice.

[0005] It has now surprisingly been found that by appropriate selectionof APP expression construct, high levels of transgene mRNA are obtained,which exceed the endogenous APP message by up to 10 fold, and result incorrespondingly elevated protein levels. Moreover, on histologicalanalysis, significant deposits of human βA4 peptide are observed.Additionally and even more importantly, hyperphosphorylation of themicrotubule-associated protein tau is achieved, which is a pathologicalphenotype associated to AD. Furthermore, the deposits accumulatecholinesterase staining associated with a local distorsion ofcholinergic fibers typically observed in AD. Both features have not beenreported previously with analogous transgenic animals. The pathology isaccompanied with selective neuron loss in distinct areas of the brain.

[0006] Accordingly in a first aspect the invention provides arecombinant DNA construct comprising a polynucleotide encoding a humanAPP polypeptide comprising the Swedish mutation, functionally linked toa Thy-1 promoter element, provided that the Thy-1 promoter element is arodent, e.g. mouse, Thy-1 promoter element when the Swedish mutation isthe only mutation present in the APP polypeptide.

[0007] Transgenic mice expressing said mutated human APP under controlof said promotor have been found to develop a pathological phenotypewhich goes beyond that previously described by Games et al. [Nature 373,523-527 (1995)], by combining APP and tau linked features-of the ADpathology. Moreover, the mice have been found to present behaviouralchanges characteristic of AD, which has also never been reported beforewith transgenic animals.

[0008] It will be appreciated that such mice, by closely reflecting theAD pathology, as well as their transgenic cells, are particularly usefulmodels of the disease.

[0009] Accordingly in a further aspect the invention provides transgenicnon-human animals which exhibit both APP and tau-linked features, e.g.histological features, of AD pathology, and preferably also behaviouralchanges characteristic of AD.

[0010] Suitably the transgenic non-human animals express a human APPcomprising the Swedish mutation or the Swedish mutation in combinationwith one or more additional mutations, in particular the Londonmutation. Suitably also the transgenic animal exhibits the features ofAD pathology before 12 months of age preferably by about 6 months ofage. Conveniently the transgenic animal is a rodent e.g. a mouse or arat, preferably a mouse. This aspect of the invention includestransgenic cells derived from the transgenic non-human animal.

[0011] Without prejudice to the generality of the present invention, itappears that the level at which the transgene is expressed in thetransgenic animal e.g. the level of transgene mRNA, is an importantfactor for obtaining AD pathology in the animal.

[0012] Thus in a further aspect the present invention provides atransgenic non-human animal cell, wherein DNA coding for a human APPhaving only one mutation is expressed at such a level that the amount oftransgene mRNA exceeds the endogenous APP message by about 5 times, e.g.from 3 to 6 times, or more, e.g. from about 5 to about 10 times, as wellas a transgenic non-human animal, e.g. a mouse or a rat, preferably amouse, in the cells of which DNA coding for a human APP having only onemutation is expressed at such a level that the amount of transgene mRNAexceeds the endogenous message by about 5 times or more.

[0013] The only one mutation present in the APP polypeptide may compriseany APP mutation, including the Swedish mutation or the London mutationor other mutations at amino acid 717. Preferably the only one mutationis the Swedish mutation.

[0014] It furthermore appears that the number of genetic lesionsinfluencing the production of βA4 introduced in a transgenic animal isanother important factor for obtaining AD pathology in the animal.

[0015] The invention also provides a transgenic non-human animal cell,wherein DNA coding for a human APP having 2 mutations is expressed atsuch a level that the amount of transgene mRNA exceeds the endogenousAPP message by about 2 times, e.g. from 1.5 to 3 times, as well as atransgenic non-human animal, e.g. a mouse or a rat, preferably a mouse,in the cells of which DNA coding for a human APP is expressed at such alevel that the amount of transgene mRNA exceeds the endogenous messageby about 2 times.

[0016] Further the invention provides a transgenic non-human animalcell, wherein DNA coding for a human APP having 3 or more mutations isexpressed at such a level that the amount of transgene mRNA exceeds theendogenous APP message by less than 2 times, e.g. from about 1 to 2times, as well as a transgenic non-human animal, e.g. a mouse or a rat,preferably a mouse, in the cells of which DNA coding for a human APP isexpressed at such a level that the amount of transgene mRNA exceeds theendogenous message by less than 2 times.

[0017] The 2 mutations or 3 or more mutations may comprise anycombination of 2 or 3 or more APP mutations. Preferably, however, suchmultiple mutations comprise a combination of the Swedish and Londonmutations.

[0018] The DNA coding for human APP may comprise cDNA and/or genomicDNA, and is conveniently cDNA.

[0019] More particularly the present invention provides a transgenicnon-human animal cell, wherein DNA encoding a human APP polypeptidecomprising the Swedish mutation is expressed under the transcriptionalcontrol of a Thy-1 promotor element, as well as a transgenic non-humananimal, e.g. a mouse or a rat, preferably a mouse, in the cells of whichDNA encoding a human APP polypeptide comprising the Swedish mutation isexpressed under the transcriptional control of a Thy-1 promotor element,provided that when the Swedish mutation is the only mutation present inthe APP polypeptide the Thy-1 promoter element is a rodent, e.g. mouse,Thy-1 promoter element.

[0020] Transgenic animals according to the invention include animalsinto which the construct has been introduced directly as well as progenyof such animals which retain the ability to express the construct.

[0021] Cells manipulated according to the invention may be prepared byany known transfection technique. The DNA sequence may be introduced bydirect genetic manipulation or into an earlier generation of the cell.Thus, the cells may be obtained from transgenic animals and cultured invitro.

[0022] Also the transgenic animals may be generated according to wellestablished methods, such as manipulation of embryos, e.g. by genetransfer into embryonic stem cells, retroviral infection of earlyembryos or pronuclear microinjection.

[0023] The pronuclear microinjection technique is preferred.Transcription units obtained from a recombinant DNA construct of theinvention are injected into pronuclei of animal embryos and the obtainedfounder transgenics are bred.

[0024] The results obtained in the offspring can be analysed usingvarious techniques well known in the art. Thus, for example, transgeneAPP mRNA expression is analysed by RNA blotting, the expression patternof the transgene in the brain is determined by in situ hybridization,detection of APP in the brain is effected using immunoblottingtechniques (western blot analysis) and the effects of the expression arestudied by histology and immunohistology.

[0025] Models based on cells and animals of the invention may be usedfor example to identify and assess the efficacy of potential therapeuticagents in neurodegenerative diseases, particularly in diseases where βA4peptide is deposited and/or the microtubule-associated protein tau ishyperphosphorylated, more particularly in AD. In particular such modelsmay be used in screening or characterization assays for detecting agentslikely to prevent βA4 deposit and/or hyperphosphorylation of tau.

[0026] Accordingly in a further aspect the invention comprises a methodfor testing a potential therapeutic agent for a specified condition, inparticular a neurodegenerative disease, preferably AD, wherein a cell ofthe invention is used as target cell. More particularly it comprisessuch a method, wherein the agent is administered to a transgenicnon-human animal of the invention. Moreover the invention comprises ascreening or characterization assay consisting in or including such amethod, as well as a screening assay kit comprising cells of theinvention.

[0027] Methods for screening potential therapeutic agents using celllines or animals are well known in the art. The cells and animals of thepresent invention may be used in analogous manner.

[0028] The recombinant cells may for example be incubated with thepotential therapeutic agent and with antibodies recognizing βA4 amyloidin typical senile and diffuse plaques and/or with tau antibodiesstaining neurofibrillary tangles in the Alzheimer brain. In methodswhere the transgenic animals themselves are used, the effects of thepotential therapeutic agent may be determined by carrying out variousinvestigations on the animals after sacrifice. Also after administrationof the potential therapeutic agent, the transgenic animal may undergobehavioural testing in order to monitor cognitive function.

[0029] The techniques of detection of βA4 and protein tau, includingWestern blot analysis, and the antibodies used therefor, are also welldocumented.

[0030] Compounds for use in the treatment of neurodegenerative diseases,which have been identified using an assay or assay kit as defined above,are also part of the present invention.

[0031] The following example illustrates the invention:

[0032] Expression Construct

[0033] Human APP751 cDNA carrying the Swedish double mutation ismodified at the 5′ end to reconstitute an optimal translation initiationsequence (GCC GCC ATG G).

[0034] This cDNA starting at above sequence and extending to nucleotide3026 (Hind III site) is inserted into the Xho I cloning site of apUC18-based vector containing an 8.1 kb EcoRI fragment comprising themouse Thy-1.2 gene [Vidal et al. (1990) EMBO J. 9, 833-840]. The vectoris modified such that a 1.5 kb BanI-Xhol fragment carrying exon 3 andflanking intervening sequences is replaced by a linker sequence encodingthe unique Xho I recognition site [Moechars et al. (1996) EMBO J. 15,1265-1274]. Transcription units are released by Notl/Pvul digestion.

[0035] Expression construct APP 14 described in K. Andrä et al.,Neurobiology of Aging, Vol. 17, No. 2, 183-190 (1996) is modified byreplacing a 600 bp Bgl II/Spe I fragment with a corresponding fragmentof a human APP₇₅₁ cDNA carrying the London mutation V 717→I.Transcription units are released by Not I digestion.

[0036] Generation of Transgenic Mice

[0037] Isolated transcription units are injected into the pronuclei ofB6D2F1×B6D2F1 embryos to generate transgenic founder animals.

[0038] Northern Blot Analysis, in situ Hybridization, Western BlotAnalysis, Histology and Immunohistology

[0039] are performed according to the methods described in K. Andra etal., Neurobiology of Aging, Vol. 17, No. 2,183-190 (1996).

[0040] Results

[0041] Offspring of the founder animals express human APP mRNA in highamounts throughout all brain structures as demonstrated by in situhybridization. Determined amounts of transgene derived protein exceedthose of endogenous APP 5 to 10 fold. At 6 months of age, these miceshow extracellular deposits of human βA4 peptide in cerebral cortex andthe hippocampal formation. These deposits are positive in methenaminesilver impregnation, thioflavin S staining and in Congo Redbirefringence. They are surrounded by reactive astrocytes and dystrophicneurites. In addition, plaques are immunoreactive with antisera specificto hyperphosphorylated microtubule associated protein tau as found inbrains of AD patients, which has not been reported previously foranalogous transgenic animals. Hence, the described deposits in thebrains of these mice closely resemble senile plaques found in ADpatients. When stained for acetylcholinesterase, a strong labelling ofplaques and a local distorsion of the cholinergic fibre nerwork isobserved. Plaques contain acetylcholinesterase activity in structuresresembling swollen, dystrophic neurites. This degeneration ofcholinergic neurites is another well-known feature associated with AD.Furthermore, a local degeneration of neurons in the plaque vicinity isobserved in areas typically affected in AD such as hippocampal CA1.Here, the neuron loss is negatively correlated to the plaque burden andcan reach up to 20%.

[0042] Tau hyperphosphorylation, cholinesterase staining and neuron lossin APP transgenic mice according to the invention are illustrated inFIG. 1. Staining of plaques with tau antibody AT8 recognizingphosphorylated Ser202 and Thr205 of tau is shown on a sagital freefloating section of a transgenic mouse brain in A and in highermagnification in D. Western blots of brain extracts from transgenicmice, 6 months (2) and 15 months (4) of age and littermate controls(1,3) are shown in B and C. Blots were stained with antibodies AT8 (B)and N-tau7 (C) recognizing tau in a phosphorylation dependent andindependent manner, respectively. Numbers indicate molecular weights ofmarker proteins in kDa. E shows staining for acetylcholine esterase intransgenic mice. A local distorsion of cholinergic fibers in the plaquevicinity can be noted. The loss of pyramidal neurons in the vicinity ofAβ deposits in area CA3 is shown in F by toluidine blue staining.

[0043] Behavioural Testing

[0044] Transgenic mice obtained as described above show significantnon-cognitive behavioural changes corresponding to changes observed withpatients suffering from AD, as reported by Mega et al. (1996) Neurology46, 130-135.

[0045] For example in the Half-Enclosed Platform test according to amodification of Käsermann (1986) Psychopharmacol. 89, 31-37, compared tonon-transgenic littermates, the animals avoided the open half and anincrease of exploratory-behavioural moves and postures such aslocomotion and head raising, indicative of agitation, disinhibition andirritability as reported for AD patients was observed.

[0046] Cognitive Testing

[0047] Furthermore the mice show significant cognitive impairment.

[0048] For example in the water maze according to Morris et al. (1982)Nature 297, 681-683, compared to non-transgenic littermates, the animalsmade significantly less crossings of the annulus representing theplatform's previous position (2.5±0.5 vs. 4.4±0.7; p<0.05, 2-tailMann-Whitney U-test) and spent a significantly lower percentage of timein the quadrant containing the annulus (20.8±3.8 vs. 33.1±3.2; p<0.05,2-tail Mann-Whitney U-test).

1. A recombinant DNA construct comprising a polynucleotide encoding ahuman APP polypeptide comprising the Swedish mutation, functionallylinked to a Thy-1 promoter element, provided that the Thy-1 promoterelement is a rodent, e.g. mouse, Thy-1 promoter element when the Swedishmutation is the only mutation present in the APP polypeptide.
 2. Arecombinant DNA construct according to claim 1, in which the APPpolypeptide additionally comprises the London mutation.
 3. A recombinantDNA construct according to claim 1 or 2, in which the Thy-1 promoterelement is a human Thy-1 promoter element.
 4. A transgenic non-humananimal which exhibits both APP and tau-linked features of AD pathology,and preferably also behavioural changes of AD, and transgenic cellsthereof.
 5. A transgenic non-human animal cell, wherein DNA encoding amutant human APP comprising only one mutation is expressed at such alevel that the amount of transgene mRNA exceeds the endogenous messageby about 5 times or more.
 6. A transgenic non-human animal cellaccording to claim 5 in which the only one mutation is the Swedishmutation.
 7. A transgenic non-human animal cell, wherein DNA encoding amutant human APP comprising two mutations is expressed at such a levelthat the amount of transgene mRNA exceeds the endogenous message byabout 2 times
 8. A transgenic non-human animal cell according to claim 7in which the two mutations are the Swedish mutation and the Londonmutation.
 9. A transgenic non-human animal cell, wherein DNA encoding amutant human APP comprising the three or more mutations is expressed atsuch a level that the amount of transgene mRNA exceeds the endogenousmessage by 2 times or less.
 10. A transgenic non-human animal cell,wherein DNA encoding a human APP polypeptide comprising the Swedishmutation is expressed under the transcriptional control of a Thy-1promoter element, provided that when Swedish mutation is the onlymutation present in the APP polypeptides the Thy-1 promoter element is arodent, e.g. mouse, Thy-1 promoter element.
 11. A transgenic non-humananimal cell according to claim 10, in which the human APP polypeptideadditionally comprises the London mutation.
 12. A transgenic non-humananimal cell according to claim 11, wherein the Thy-1 promoter element isa human Thy-1 promoter element.
 13. A transgenic non-human animal, inthe cells of which DNA encoding a human APP having only one mutation isexpressed at such a level that the amount of transgene mRNA exceeds theendogenous message by about 5 times or more.
 14. A transgenic non-humananimal according to claim 13, in which the only one mutation is theSwedish mutation.
 15. A transgenic non-human animal, in the cells ofwhich DNA encoding a human APP having two mutations is expressed at sucha level that the amount of transgene mRNA exceeds the endogenous messageby about 2 times.
 16. A transgenic non-human animal according to claim15, in which the two mutations are the Swedish mutation and the Londonmutation.
 17. A transgenic non-human animal, in the cells of which DNAencoding a human APP polypeptide comprising the Swedish mutation isexpressed under the transcriptional control of a Thy-1 promoter element,provided that when the Swedish mutation is the only mutation present inthe APP polypeptide the Thy-1 promoter element is a rodent, e.g. mouse,Thy-1 promoter element.
 18. A transgenic non-human animal according toclaim 17 in which the APP polypeptide additionally comprises the Londonmutation.
 19. A transgenic non-human animal according to claim 13 to 18,which is a mouse.
 20. A method of producing a transgenic non-humananimal, wherein said animal is generated by incorporating a recombinantDNA construct according to claim 1 into its genome.
 21. A method ofproducing transgenic non-human animals capable of developing aneurodegenerative disease pathology, comprising injection oftranscription units obtained from a recombinant DNA construct accordingto claim 1 into pronuclei of non-human animal embryos and breeding theso obtained founder animals.
 22. A method for testing a potentialtherapeutic agent for a specified condition, wherein a transgenic animalaccording to any one of claims 13 to 19 is used or a cell according toclaim 5 to 12 is used as target cell.
 23. A method according to claim22, wherein the agent is administered to a transgenic non-human animalproduced according to the method of claim 20 or
 21. 24. A methodaccording to claim 22, wherein the condition is a neurodegenerativedisease.
 25. A method according to claim 22, wherein the condition isAlzheimer's disease.
 26. A screening or characterization assayconsisting in or including a method according to any one of claims 23 to25.
 27. A screening assay kit comprising cells according to any one ofclaims 5 to
 12. 28. A compound for use in the treatment of aneurodegenerative disease, which has been identified using an assay orassay kit according to claim 26 or 27.